Nucleotide incorporation by human DNA polymerase opposite benzopyrene
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《核酸研究医学期刊》
Laboratory of Molecular Genetics, National Institute of Environmental Health Sciences, National Institutes of Health, DHHS, PO Box 12233, Research Triangle Park, NC 27709, USA and 1 Laboratory of Bioorganic Chemistry, National Institute of Diabetes, Digestive and Kidney Diseases, National Institutes of Health, DHHS, Bethesda, MD 20892, USA
*To whom correspondence should be addressed. Tel: +1 919 541 4792; Fax: +1 919 541 7613; Email: copelan1@niehs.nih.gov
Permanent address:
Maria A. Graziewicz, Institute of Biochemistry and Biophysics, Polish Academy of Science, 02-106 Warsaw, Poland
ABSTRACT
Mitochondria are major cellular targets of benzopyrene (BaP), a known carcinogen that also inhibits mitochondrial proliferation. Here, we report for the first time the effect of site-specific N2-deoxyguanosine (dG) and N6-deoxyadenosine (dA) adducts derived from BaP 7,8-diol 9,10-epoxide (BaP DE) and dA adducts from benzophenanthrene 3,4-diol 1,2-epoxide (BcPh DE) on DNA replication by exonuclease-deficient human mitochondrial DNA polymerase (pol ) with and without the p55 processivity subunit. The catalytic subunit alone primarily misincorporated dAMP and dGMP opposite the BaP DE–dG adducts, and incorporated the correct dTMP as well as the incorrect dAMP opposite the DE–dA adducts derived from both BaP and BcPh. In the presence of p55 the polymerase incorporated all four nucleotides and catalyzed limited translesion synthesis past BaP DE–dG adducts but not past BaP or BcPh DE–dA adducts. Thus, all these adducts cause erroneous purine incorporation and significant blockage of further primer elongation. Purine misincorporation by pol opposite the BaP DE–dG adducts resembles that observed with the Y family pol . Blockage of translesion synthesis by these DE adducts is consistent with known BaP inhibition of mitochondrial (mt)DNA synthesis and suggests that continued exposure to BaP reduces mtDNA copy number, increasing the opportunity for repopulation with pre-existing mutant mtDNA and a resultant risk of mitochondrial genetic diseases.
INTRODUCTION
Polycyclic aromatic hydrocarbons (PAHs), many of which are carcinogenic, are widespread environmental contaminants produced by incomplete combustion. Mammalian metabolism of carcinogenic PAHs mediated by cytochromes P450 and epoxide hydrolase generates, among other metabolites, bay region diol epoxides (DE). The tumorigenic potential of these metabolites (1) most likely results from their reaction with cellular DNA to form miscoding adducts. Two diastereomers of a given bay region DE are metabolically possible, one in which the benzylic hydroxyl group and the epoxide oxygen are cis and one in which these two groups are trans. In addition, each of these diastereomers exists as a pair of enantiomers. In the present study, only DNA adducts derived from the pair of enantiomers of the DE diastereomer with the trans orientation of the benzylic hydroxyl and epoxide oxygen (Fig. 1A) are used. Of the metabolically possible DEs from a given hydrocarbon, the (R,S,S,R)-DE (Fig. 1A) is both metabolically predominant and the most carcinogenic (2–4). The principal sites of DNA adduct formation are at the exocyclic N2 and N6 amino groups of deoxyguanosine (dG) and deoxyadenosine (dA), respectively (5). PAH DE adducts in DNA induce mutations in both mammalian cells (6–8) and bacteria (6,7,9,10), thus providing an attractive mechanism for the initiation of cell transformation.
Figure 1. (A) Structures of the benzopyrene (BaP) and benzophenanthrene (BcPh) diol epoxide (DE) enantiomers (diastereomer with a benzylic hydroxyl group and epoxide oxygen trans), as well as their DNA adducts used in this study (dG = dG residue and dA = dA residue with the point of attachment to the hydrocarbon at N2 and N6, respectively). Note that the configuration at the benzylic carbon of the epoxide is inverted on trans opening of the epoxide and retained on cis opening. (B) Template–primer substrates for pol with dG and dA adducts at the indicated positions (G* and A*, respectively). A 12mer complementary to the 5'-end of each 22mer primer was annealed to the primer in order to create a substrate with a 22mer primer and a 26mer template containing a gap that does not affect its enzymatic processing. Controls consisted of substrates with the normal (unadducted) bases at positions G* and A*.
Mitochondrial DNA (mtDNA) has long been suspected to be a major cellular target of chemical carcinogens (11–19). Benzopyrene (BaP) has been found to localize specifically in mitochondria (13). Several studies have demonstrated that mtDNA suffers more modification than nuclear DNA after exposure of cells to BaP or BaP DE (11,20). In isolated, whole mitochondria from BaP-treated rats, DNA synthesis is reduced but mitochondrial DNA polymerase activity increases, most likely as a feedback response to the high degree of mtDNA modification and decrease in ATP production (16). Treatment of cells with BaP inhibits mitochondrial DNA synthesis with the associated presence of 4–15 times more adducts per unit length of mtDNA than nuclear DNA (21). Recently, using a highly sensitive chemiluminescence immunoassay, levels of BaP DE adducts at dG were found to be 10-fold higher in mtDNA than nuclear DNA per unit length of DNA (22). Antioxidant anticancer treatment with N-acetylcysteine decreased the amount of mtDNA adducts in rats exposed to cigarette smoke (23), which contains PAH carcinogens, possibly by trapping the reactive DE metabolites. Thus, PAHs may exert a more profound effect in the cell on mtDNA relative to nuclear DNA. This may be due to the lipophilic character of these PAHs and the high ratio of lipid to DNA in mitochondria.
Mitochondrial DNA is replicated by an assembly of proteins and enzymes consisting of polymerase (pol) , single-stranded DNA binding protein, DNA helicase and a number of accessory proteins and transcription factors (24,25). In humans, pol consists of two subunits, a 140 kDa catalytic subunit containing DNA polymerase, exonucleolytic proofreading and dRP lyase activities and a smaller 55 kDa subunit that is a processivity factor with the ability to bind double-stranded DNA (26–28).
In the present study, translesion synthesis on DNA templates containing BaP or benzophenanthrene (BcPh) DE adducts by human recombinant pol was examined. We used oligonucleotide substrates, one in which a guanine (G) is modified and the other in which an adenosine (A) is modified, which had previously been examined in both mutagenesis (9) and polymerase bypass (29,30) studies. The human pol was able to incorporate correct and incorrect nucleotides opposite most of these adducts. The BaP and BcPh adducts caused significant blockage of replication with limited translesion synthesis past certain stereoisomers.
MATERIALS AND METHODS
Enzymes
The exonuclease-deficient form of human DNA pol (Exo–p140) was purified to homogeneity from baculovirus-infected insect cells as described (26). The recombinant human accessory subunit (p55) was purified to homogeneity from Escherichia coli and the heterodimeric forms of the polymerase were reconstituted as previously described (28).
Oligonucleotides
The G* templates containing a single trans-opened BaP DE trans R or trans S dG adduct (Fig. 1A) on the fourth nucleotide from 5'-end in the sequence context 5'-TTCG*AATCC TTCCCCC-3' (Fig. 1B) were synthesized as described previously (31,32). A* templates containing dA adducts (Fig. 1A), also on the fourth nucleotide from the 5'-end, in the sequence context 5'-CAGA*TTTAGAGTCTGC-3' (Fig. 1B) were synthesized as described: trans-opened BaP DE trans R or trans S dA adducts (33), cis-opened BaP DE cis R or cis S dA adducts (34) and cis-opened BcPh cis R or cis S dA adducts (35). All other oligonucleotides were purchased from Invitrogen Life Technologies, desalted and PAGE purified. The dG primer (5'-TCG ATC GAT CCA GGG GGA AGG A-3') for the G* templates was gel purified and labeled at the 5'-end with ATP by T4 polynucleotide kinase (36). The dG primer, the G* template containing either the BaP trans R or trans S dG adduct and a 12mer (5'-TGG ATC GAT CGA-3') were annealed in a 1:1.4:1.4 ratio to produce the substrate in Figure 1B. To construct substrates of suitable length, each adducted 16mer template and an additional 12mer were annealed to a 22mer primer (see Fig. 1B). Annealing was performed by heating the oligonucleotides to 90°C for 5 min followed by slow cooling to room temperature in 10 mM Tris–HCl, pH 7.5. Likewise, the dA primer (5'-TCG ATC GAT CCA GCA GAC TCT A-3') for the A* templates was gel purified and labeled at the 5'-end with ATP by T4 polynucleotide kinase. As above, the dA primer, each of the A* templates and the 12mer (5'-TGG ATC GAT CGA-3') were annealed in a 1:1.4:1.4 ratio to produce the substrates in Figure 1B. Control reactions for each set contained identical oligonucleotides except that the 16mer templates contained a normal dA or dG residue in place of the adducts.
DNA polymerase reactions
DNA pol reactions (10 μl final volume) contained final concentrations of 25 mM HEPES–KOH (pH 7.5), 5 mM MgCl2, various concentrations of the four dNTPs, 2 mM 2-mercaptoethanol, 50 μg/ml bovine serum albumin, 0.1 mM EDTA, 1 pmol 5'-32P-labeled 22mer previously annealed to 1.4 pmol 16mer template and 1.4 pmol 12mer (Fig. 1B). Reactions were started by addition of DNA pol (10 fmol for control substrates and 60 fmol for adducted substrates), either Exo–p140 alone or in complex with the accessory subunit (1.0:1.1 molar ratio). Samples were incubated for 5 min at 37°C. Reactions were stopped by addition of 10 μl of 95% aqueous formamide containing 25 mM EDTA and gel sequencing dye solution, followed by brief heating in a boiling water bath.
Incorporation analysis
Reaction products (4 μl) were loaded onto 15% polyacrylamide gels containing 7 M urea. Following electrophoresis, the gels were dried, exposed to a PhosphorImager screen and analyzed in a Storm 860 PhosphorImager (Molecular Dynamics). Radioactive bands were quantified with NIH Image software (version 1.62). Vmax and Km values were calculated using a non-linear Michaelis–Menten kinetic fit program from M.F. Goodman (USC). Misincorporation efficiencies were calculated from relative Vmax/Km values.
RESULTS
Human mitochondrial DNA pol has been tested for its ability to catalyze extension bypass of dG and dA adducts derived from two PAHs, as well as the specificity of nucleotide insertion opposite them using 22mer primer/28mer template complexes (see Materials and Methods). Structures of the adducts studied are shown in Figure 1A. These adducts are the BaP trans R and trans S dG adducts (in G* template–primer sets; Fig. 1B), the BaP trans R, trans S, cis R and cis S dA adducts and the BcPh cis S and cis R dA adducts (in A* template–primer sets). These adducts were copied by the exonuclease-deficient form of pol (Exo–p140) alone as well as in the complex with the pol accessory subunit, p55.
We first attempted to extend 10mer oligonucleotide primers annealed to the 16mer adducted and control templates. However, pol did not extend these short 16/10mer template–primers efficiently (<10% of primers extended), consistent with a requirement by the enzyme for a longer piece of DNA for optimal binding. We therefore synthesized a longer 22mer primer as well as a 12mer template extension and assembled the oligonucleotides as shown in Figure 1B. These longer template–primer sets were >3-fold more efficiently extended compared to the 10mer primers. We performed primer extension reactions with oligonucleotides in a running start reaction to reflect more accurately processive replication. These running start reactions were initiated by synthesis opposite either two T or two A residues in the template with a low concentration (1 μM) of either dATP or dTTP, respectively. The low dose of either dATP or dTTP was supplied to progress the polymerase to the site of the adduct. Incorporation opposite the adduct was tested by addition of each of the nucleoside triphosphates at high concentration. At least 1.0 mM dNTP was needed in order to detect incorporation opposite most of the adducts studied.
Pol -dependent replication of two diastereomeric BaP DE–dG adducts with and without the pol accessory subunit
Translesion synthesis by human pol with and without the accessory subunit was tested with the trans R and trans S diastereomers of the BaP DE–dG adducts using the first template–primer set in Figure 1B. As can be seen in lane 3 in each panel of Figure 2, the low concentration of dTTP was sufficient to fill the 2 nt gap prior to the site of the adduct, but did not cause any further incorporation or misincorporation opposite the adducts or the control template G. Addition of each nucleotide separately at high concentration is depicted in lanes 4–7 (Fig. 2A, Exo– p140 alone). With 1 mM each nucleoside triphosphate, pol easily incorporated the correct as well as the incorrect nucleotides on the control template. With the isolated catalytic subunit we even observed misincorporation of dGMP and extension of this misincorporation (lane 7 for no lesion), as well as incorrect elongation after a correctly incorporated dCMP residue (lane 6).
Figure 2. Single base insertion opposite normal dG and BaP trans R and trans S dG adducts in oligonucleotides. Reactions containing either Exo–p140 (A) or Exo–p140/p55 complex (B) were incubated for 5 min at 37°C. A partial sequence of the G* templates (Fig. 1B) is shown vertically on both panels of the figure, with the BaP-modified guanine indicated as G**. Lane 0, no dNTPs added; lane 4, all four dNTPs (each at 1 mM final concentration) added; lane T*, 1 μM dTTP (added to initiate a running start one base before the BaP-adducted guanine, G**); lanes A, T, C and G, 1 μM dTTP plus 1 mM one of the four dNTPs. The arrow (B) indicates the products of extension past the adduct.
The scenario for the action of pol on the substrates containing BaP DE–dG adducts was quite different. Here, pol incorporated mainly dAMP opposite the BaP DE adducts (Fig. 2A, lane 4), with a lesser efficiency of dGMP incorporation (Fig. 2A, lane 7). No correct incorporation of dCMP or incorrect incorporation of dTMP opposite these adducts was observed with the p140 subunit. The ability of the polymerase to extend past these adducts was tested by addition of all four dNTPs (lane 2 of each panel). As expected, the polymerase was easily able to extend synthesis to the end of the non-adducted template (Fig. 2A). Addition of all four dNTPs to reactions containing the BaP DE–dG adducted templates resulted in incorporation of only a single nucleotide residue opposite the adduct, similar to the results observed with either dGTP or dATP alone. Thus, the isolated catalytic subunit misincorporated either a dGMP or dAMP opposite the trans R and trans S adducts but could not extend these products.
Addition of the accessory p55 subunit (Fig. 2B) caused the polymerase to synthesize more product with the non-adducted template as well as with the BaP trans R and trans S dG adducted templates. With all four nucleotides present, pol with the accessory subunit inserted one or more nucleotides past the end of the template, most likely through a template dislocation intermediate or strand slippage mechanism that we previously documented for pol (37). The Exo– p140/p55 complex was able to incorporate all four nucleotides and extended past the resulting (mis)pair by at least one additional dAMP, dCMP or dGMP nucleotide on the control template with 1 mM each dNTP. With the BaP dG adducts, the p55 accessory subunit caused the polymerase to incorporate all of the nucleotides opposite the BaP DE–dG adducts, as compared to just dGMP or dAMP incorporation with the isolated catalytic subunit. In addition, a 1 nt translesion extension was observed with either 1 mM dATP or dGTP (Fig. 2B, lanes 4 and 7, arrow). In the presence of all four dNTPs (lane 2), the presence of p55 resulted in the 1 nt extension observed with either dATP or dGTP alone but failed to cause further extension.
Pol -dependent replication of four diastereomeric BaP DE–dA adducts with and without the pol accessory subunit
Translesion synthesis opposite BaP trans S, BaP trans R, BaP cis S and BaP cis R dA adducts was tested in a different sequence context, using the running start A* template–primer sets shown in Figure 1B. In these template–primer sets a low concentration of dATP was required to progress the polymerase to the site of the adduct in a processive synthesis. With the control template no significant amount of misincorporation of 1 μM dAMP by either the catalytic subunit alone or the complex containing the accessory subunit was observed (Fig. 3A and B, control template, lanes 3). With the addition of each of the four nucleoside triphosphates at high concentration (1 mM) to the control template we observed not only incorporation of the cognate dTMP into the primer but also the misincorporation of dAMP and dCMP. In addition extension of the A-C mispair by correct incorporation of dCMP opposite the next G was observed. Complete extension was observed when high concentrations of all four dNTPs were present. Qualitatively, the results were the same with and without the accessory subunit present.
Figure 3. Single base insertion opposite normal dA and BaP trans R, cis R, trans S and cis S dA adducts in oligonucleotides. Reactions containing either Exo–p140 (A) or Exo–p140/p55 complex (B) were incubated for 5 min at 37°C. A partial sequence of the A* templates (Fig. 1B) is shown vertically on both panels of the figure, with the BaP-modified adenine indicated as A**. Concentrations of the dNTPs and lane designations are as in Figure 2, except that each incubation contained 1 μM dATP to initiate a running start on the A* templates.
With the higher amount of pol needed to detect incorporation opposite the BaP DE–dA adduct we observed a slight amount of dAMP incorporation in the presence of the low 1 μM dATP needed to progress the polymerase to the site of the adduct. This amount of misincorporation with 1 μM dATP varied depending on adduct stereochemistry, but was generally low enough not to interfere with interpretation of the incorporation of the other nucleotides. In the presence of 1 mM dATP this misincorporation of dAMP opposite all four of the adducts was significantly increased. At high concentration the only other nucleotide to show significant incorporation relative to the background of dAMP was the cognate dTMP in the case of the BaP trans R and trans S dA adducts, while no significant dTMP incorporation was observed with the BaP cis R and cis S dA adducts. No dCMP or dGMP incorporation was observed with any of the BaP dA adducts. With all four nucleotides present only trace amounts of extension were observed on the BaP trans R and trans S dA templates and extension was not detected on the BaP cis R and cis S dA templates.
Upon addition of the p55 accessory subunit to these reactions, we observed enhanced incorporation with all of the BaP DE–dA adducts. The accessory subunit allowed correct incorporation of dTMP opposite the BaP trans R and trans S dA adducts, to about the same extent as the misincorporation of dA. There was no evidence for enhanced misincorporation of dCMP or dGMP opposite the trans adducts, since these bands (Fig. 3B) were the same as those observed with 1 μM dATP alone. The situation with the BaP cis R and cis S dA adducted templates was very different in the presence of the accessory subunit. Here, we observed primarily dAMP misincorporation as well as slight incorporation of dCMP and possibly dGMP, which were not observed in the absence of the p55 subunit. There was little or no enhanced incorporation of dTMP opposite the adduct in the presence of the accessory subunit. Thus the effect of the accessory subunit on incorporation opposite BaP DE–dA adducts varies with the stereoisomer.
Pol -dependent replication of two stereoisomeric BcPh DE–dA adducts with and without the pol accessory subunit
Incorporation and translesion synthesis on a running start template having the same sequence as that used with the BaP dA adducts (Fig. 1B) were also examined with BcPh cis R and cis S dA adducts. The incorporation by pol with and without the accessory subunit on the non-adducted template is shown as the control in Figure 4A and B, respectively. With the catalytic subunit alone, dAMP was the only nucleotide incorporated opposite either diastereomeric BcPh dA adduct, with more dAMP being misincorporated opposite the cis S diastereomer (Fig. 4A, lane 4). In the presence of all four dNTPs, incorporation (presumably of dA) opposite the adduct occurred, but no further translesion synthesis was observed (Fig. 4A, lane 2).
Figure 4. Single base insertion opposite normal dA and BcPh cis R and cis S dA adducts in oligonucleotides. Reactions containing either Exo–p140 (A) or Exo–p140/p55 complex (B) were incubated for 5 min at 37°C. A partial sequence of the A* template is shown vertically on both panels of the figure, with the BcPh-modified adenine indicated as A**. Concentrations of the dNTPs and lane designations are as in Figure 3.
Addition of the p55 accessory subunit to the BcPh-adducted template reactions allowed nucleotides other than dAMP to be incorporated. With the cis R diastereomer, all four nucleotides were incorporated. When all four dNTPs were present together, the pol complex was able to incorporate a small amount of nucleotide one base past the adduct, but no further extension was detected. With the BcPh cis S isomer, dCMP and dGMP but not the cognate dTMP were incorporated by the pol complex, and no further extension was detected.
Kinetics of nucleotide insertion opposite BaP and BcPh adducts by pol
To determine relative efficiency of incorporation and misincorporation by pol opposite the adducts, we performed kinetic analysis of primer extension reactions. As in our other experiments, exonuclease-deficient pol was used to avoid degradation of the primer by the proofreading activity and to simplify interpretation of the results. This strategy was imperative due to the relatively high amounts of enzyme required to detect incorporation opposite the adducts. With the G* template–primer sets, the running start reaction requires processive DNA synthesis by inserting two dTMPs (I1 and I2) prior to the target G* site for the correct or incorrect nucleotide (I3). From integration of the intensities of PhosphorImager scans of I1, I2 and I3, reaction velocities were determined and misinsertion efficiency (fins) was calculated as the ratio Vmax/Km for incorrect and correct nucleotides. Velocity was expressed as the ratio I3/I2 as described (38,39). The same approach used dATP to accomplish the running start with the A* template–primer sets (Fig. 1B).
For the kinetic studies with G* template–primer sets, the concentration of dTTP required to fill the I1 and I2 positions was lowered to minimize the misinsertion of dTMP opposite the dG adduct that we observed in Figure 2. Since incorporation opposite these adducts was very inefficient in the absence of the accessory subunit, only the kinetic parameters for the two-subunit complex are shown. Table 1 displays the apparent kinetic parameters for incorporation opposite the BaP trans S and trans R dG adducts. Steady-state assumptions allow the overall efficiency of each enzyme on a normal DNA template to be estimated as kcat/Km(dNTP), a parameter comparable to the pre-steady-state indicator of enzymatic efficiency kpol/Kd(dNTP) (40,41). With saturating template–primer concentrations the ratio kcat/Km(dNTP) does not change upon restriction of DNA synthesis (42), which is encountered by replication opposite damaged template bases, and kcat/Km(dNTP) remains a valid parameter for comparing insertion events by pol . Similarly, since steady-state parameters do not account for the expected differences in Kd(dNTP) with normal or damaged templates, comparisons of differential insertion by a given enzyme are best described as fins = (Vmax/Km)incorrect/(Vmax/Km)correct.
Table 1. Kinetics of insertion by the p140/p55 pol complex opposite the BaP trans R and BaP trans S dG adducts in the sequence 3'-...AAG*CTT-5'
Individual kinetic parameters are presented in the tables as Vmax/Km(dNTP) ratios and fins values. The kinetic values for the BaP trans R and trans S dG adducts (Table 1) corroborate the observations in Figure 2. Although fidelity of the exonuclease-deficient pol in the presence of the p55 accessory subunit on the non-adducted dG substrate was low, presumably due to the effects of the accessory subunit, the correct dCMP was still much more effectively inserted opposite a normal dG base than opposite either of the adducts. Incorporation of dCMP opposite either dG adduct was 200 000- to 400 000-fold less efficient than dCMP incorporation opposite the non-adducted template dG bases (compare Vmax/Km values between the non-adducted template and BaP-adducted templates). In fact, dCMP was the worst nucleotide incorporated, while dAMP and dGMP were most efficiently incorporated opposite the BaP dG adducts. The fins values indicate that dAMP was incorporated 4.3 to 4.7 times more readily than dCMP on the adducted templates, whereas dAMP was incorporated 100-fold less efficiently than dCMP on the non-adducted template. Pol showed little difference in incorporation efficiency of each nucleotide between the trans R and trans S isomers of the BaP DE–dG adducts (Table 1).
In contrast, the kinetics of nucleotide incorporation opposite the BaP DE–dA adducts by pol clearly revealed differences between the adduct stereoisomers (Table 2), especially in incorporation of dGMP and dCMP between the cis and trans adducts. With either the BaP trans R or trans S dA adduct we observed no detectable (>1% extended product) incorporation of dGMP or dCMP, whereas with either the cis R or cis S dA adduct we could readily detect and quantify the level of dGMP and dCMP incorporation. However, there was little difference between the BaP cis R and cis S dA adducts or between the BaP trans R and trans S dA adducts. Incorporation of the cognate dTMP opposite the BaP DE–dA adducts was between 7500- and 41 000-fold less efficient than dTMP incorporation on the non-adducted template. Incorrect dAMP was the most efficiently incorporated nucleotide opposite all four of the isomeric BaP DE–dA adducts, although its incorporation was still less efficient than that for dAMP misincorporation opposite the non-adducted dA template. As in the case of the dG adducts, incorporation of the correct nucleotide was more seriously hindered by an adduct than incorporation of an erroneous dAMP, resulting in increased misincorporation frequencies opposite the adducts.
Table 2. Kinetics of insertion by the p140/p55 pol complex opposite the BaP trans R, BaP trans S, BaP cis R and BaP cis S dA adducts in the sequence 3'-...TTA*GAC-5'
Incorporation opposite the BcPh cis R and cis S dA adducts (Fig. 1) was detected with each of the four dNTPs (Table 3). Vmax/Km ratios were quite similar for the BcPh cis dA adducts and their corresponding BaP analogs (within a factor of 3 or less except for dTMP incorporation opposite the BcPh cis R adduct, which was 6 times less efficient than opposite the corresponding BaP cis R adduct). Incorporation of the cognate dTMP was 106 000- and 41 000-fold less efficient on the BcPh cis R and cis S dA templates as compared to the non-adducted dA template, whereas misincorporation of dAMP on both BcPh templates was only 6- to 8-fold less efficient than dAMP misincorporation on the non-adducted control template. Incorrect dAMP was 40- and 9-fold more likely to be incorporated on the BcPh cis R and cis S templates, respectively, as compared to dTMP. This was in contrast to the control template, where dAMP is incorporated more than 300 times less efficiently than dTMP by pol . Among all the adduct-templated (mis)incorporations measured in this study, misincorporations of dAMP opposite the BcPh and BaP cis dA adducts were most efficient.
Table 3. Kinetics of insertion by the p140/p55 pol complex opposite the BcPh cis R and BcPh cis S dA adducts in the sequence 3'-...TTA*GAC-5'
DISCUSSION
Reports of translesion synthesis past DNA adducts by pol are limited. The ability of pol to synthesize past abasic sites has been studied, and the enzyme was found to stall the majority of the time (43). When pol does perform translesion synthesis past an abasic site, it prefers to incorporate dAMP opposite the site (43). Translesion synthesis by pol is readily accomplished opposite 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) template bases. Xenopus laevis pol incorporates dCMP 73% of the time while misincorporating dAMP 27% of the time opposite an 8-oxo-dG adduct (43). Translesion DNA synthesis past Pt–DNA adducts by human mitochondrial DNA polymerase has been demonstrated (44). Human pol displays a specificity for translesion synthesis past dien Pt–DNA adducts, with less synthesis past oxaliplatin and still less for cisplatin (44). While damage to mtDNA by BaP is well established (11,20–23,45), translesion synthesis past BaP adducts by pol has not previously been investigated. Here we have studied base incorporation and translesion synthesis opposite BaP DE–dG, BaP DE–dA and BcPh DE–dA adducts in DNA by human mitochondrial DNA polymerase with and without the p55 accessory subunit.
The catalytic subunit alone had weak ability to incorporate opposite all of the DE adducts studied and showed no translesion synthesis. Even in the presence of the p55 accessory subunit we observed only limited, one base extension beyond the BaP DE–dG adducts, and even weaker extension or no extension at all beyond dA adducts. For all the adducts studied, kinetics experiments in the presence of p55 indicated that the total incorporation of both correct and incorrect nucleotides opposite these adducts was decreased by 3–4 orders of magnitude relative to the unmodified control template. Thus these adducts present a substantial block to DNA replication in vitro. This blockage is consistent with the inhibition of mtDNA replication that was previously demonstrated with intact mitochondria upon treatment with BaP (16,21).
Pol containing its associated 3'5' proofreading exonuclease activity is highly accurate (37). The present study required use of exonuclease-deficient pol in order to avoid degradation of the primers, resulting in markedly reduced fidelity of replication for unmodified DNA. However, even in the absence of proofreading ability, pol still inserted predominantly the correct nucleotide opposite a normal dG or dA, with an error frequency of <10–3 for most cases. In contrast, pol with its accessory subunit incorporated all three incorrect nucleotides in preference to the correct dCMP opposite dG adducts. The nucleotides most frequently inserted opposite a dG adduct were the purine nucleotides dGMP and dAMP, with fins values of 3.7–4.7 for both nucleotides. This was a consequence of a dramatically reduced incorporation of the correct dCMP along with much smaller decreases in incorporation of the incorrect nucleotides. A similar effect was observed with the dA-adducted templates, with dAMP being incorporated in preference to the correct dTMP opposite all the dA adducts studied.
Pol is a family A DNA polymerase which includes such well-studied DNA polymerases as the E.coli pol I and T7 DNA polymerase. Thus, one might expect pol to exhibit the same pattern of translesion bypass with BaP DE adducts as E.coli pol I and T7 DNA polymerase. Studies with the Sequenase 2.0 version of T7 DNA polymerase demonstrated that BaP DE–dG adducts blocked base insertion opposite the adducts in vitro (46). On the basis of molecular modeling studies, preferential dAMP insertion opposite a BaP trans S dG adduct by the T7 DNA polymerase has been postulated to result from stabilizing interactions between the polymerase, DNA and dATP (47). With dA adducts, Sequenase was also shown to be very inefficient at nucleotide incorporation and, like pol , incorporated mostly an incorrect dAMP opposite both the trans R and trans S adducts (33). The response of A family pol to adducts is also reminiscent of that of the Y family bypass polymerase , which showed a strong preference for purine nucleotide misincorporation opposite BaP DE–dG adducts (30).
Mutations in mtDNA can induce a multitude of severe metabolic disorders and mitochondrial genetic disorders (48). Although translesion synthesis by pol in the present study was inefficient and was limited to a single base extension beyond BaP DE–dG adducts, our observation of enhanced misincorporation opposite the adducts suggests a mechanism for mutagenesis, should pol be able to replicate past these adducts in vivo. Another attractive, although indirect, mechanism exists for delayed phenotypic expression of mutations as a result of DNA damage induced by PAHs. Mitochondrial DNA is a multicopy genome, with as many as 10 000 mtDNA genomes per cell, and can exist in heteroplasmic form in which a low percentage of the genomes may contain mutations, either silent or deleterious. Depending on the tissue type, deleterious mutations in mtDNA can exist below a clinical threshold in which phenotypic symptoms do not show. Increasing the percentage of the genomes which carry such mutations above the respiratory threshold of that tissue causes tissue degeneration and mitochondrial disease. Although BaP damage to mtDNA is well established (11,20) and later studies have supported this finding (21–23,45), long-term mutagenicity of this damage to mtDNA has not been observed (49,50). This may be due to the fact that most of the adducted mitochondrial genomes cause a complete block in DNA replication and loss of the damaged genomes, consistent with the observation of a decrease in mtDNA replication and mtDNA copy number after BaP treatment without accompanying fixed mutations. Thus, the multicopy nature of mtDNA gives the cell the advantage of dispensing with damaged genomes instead of attempting to repair them. However, blockage of mtDNA replication and subsequent depletion in mtDNA may have another consequence on mtDNA heteroplasmy. Replication of the remaining mitochondrial genomes would amplify any mutations (from whatever source) in this surviving population. Thus, deleterious long-term consequences of adducts from BaP and other PAHs in mtDNA could result from pre-existing mutations which reach a physiologically significant level only after the mtDNA population has been depleted by PAH adduct-related DNA damage.
ACKNOWLEDGEMENTS
We thank Drs Matthew Longley and Kasia Bebenek for critical reading of this manuscript.
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*To whom correspondence should be addressed. Tel: +1 919 541 4792; Fax: +1 919 541 7613; Email: copelan1@niehs.nih.gov
Permanent address:
Maria A. Graziewicz, Institute of Biochemistry and Biophysics, Polish Academy of Science, 02-106 Warsaw, Poland
ABSTRACT
Mitochondria are major cellular targets of benzopyrene (BaP), a known carcinogen that also inhibits mitochondrial proliferation. Here, we report for the first time the effect of site-specific N2-deoxyguanosine (dG) and N6-deoxyadenosine (dA) adducts derived from BaP 7,8-diol 9,10-epoxide (BaP DE) and dA adducts from benzophenanthrene 3,4-diol 1,2-epoxide (BcPh DE) on DNA replication by exonuclease-deficient human mitochondrial DNA polymerase (pol ) with and without the p55 processivity subunit. The catalytic subunit alone primarily misincorporated dAMP and dGMP opposite the BaP DE–dG adducts, and incorporated the correct dTMP as well as the incorrect dAMP opposite the DE–dA adducts derived from both BaP and BcPh. In the presence of p55 the polymerase incorporated all four nucleotides and catalyzed limited translesion synthesis past BaP DE–dG adducts but not past BaP or BcPh DE–dA adducts. Thus, all these adducts cause erroneous purine incorporation and significant blockage of further primer elongation. Purine misincorporation by pol opposite the BaP DE–dG adducts resembles that observed with the Y family pol . Blockage of translesion synthesis by these DE adducts is consistent with known BaP inhibition of mitochondrial (mt)DNA synthesis and suggests that continued exposure to BaP reduces mtDNA copy number, increasing the opportunity for repopulation with pre-existing mutant mtDNA and a resultant risk of mitochondrial genetic diseases.
INTRODUCTION
Polycyclic aromatic hydrocarbons (PAHs), many of which are carcinogenic, are widespread environmental contaminants produced by incomplete combustion. Mammalian metabolism of carcinogenic PAHs mediated by cytochromes P450 and epoxide hydrolase generates, among other metabolites, bay region diol epoxides (DE). The tumorigenic potential of these metabolites (1) most likely results from their reaction with cellular DNA to form miscoding adducts. Two diastereomers of a given bay region DE are metabolically possible, one in which the benzylic hydroxyl group and the epoxide oxygen are cis and one in which these two groups are trans. In addition, each of these diastereomers exists as a pair of enantiomers. In the present study, only DNA adducts derived from the pair of enantiomers of the DE diastereomer with the trans orientation of the benzylic hydroxyl and epoxide oxygen (Fig. 1A) are used. Of the metabolically possible DEs from a given hydrocarbon, the (R,S,S,R)-DE (Fig. 1A) is both metabolically predominant and the most carcinogenic (2–4). The principal sites of DNA adduct formation are at the exocyclic N2 and N6 amino groups of deoxyguanosine (dG) and deoxyadenosine (dA), respectively (5). PAH DE adducts in DNA induce mutations in both mammalian cells (6–8) and bacteria (6,7,9,10), thus providing an attractive mechanism for the initiation of cell transformation.
Figure 1. (A) Structures of the benzopyrene (BaP) and benzophenanthrene (BcPh) diol epoxide (DE) enantiomers (diastereomer with a benzylic hydroxyl group and epoxide oxygen trans), as well as their DNA adducts used in this study (dG = dG residue and dA = dA residue with the point of attachment to the hydrocarbon at N2 and N6, respectively). Note that the configuration at the benzylic carbon of the epoxide is inverted on trans opening of the epoxide and retained on cis opening. (B) Template–primer substrates for pol with dG and dA adducts at the indicated positions (G* and A*, respectively). A 12mer complementary to the 5'-end of each 22mer primer was annealed to the primer in order to create a substrate with a 22mer primer and a 26mer template containing a gap that does not affect its enzymatic processing. Controls consisted of substrates with the normal (unadducted) bases at positions G* and A*.
Mitochondrial DNA (mtDNA) has long been suspected to be a major cellular target of chemical carcinogens (11–19). Benzopyrene (BaP) has been found to localize specifically in mitochondria (13). Several studies have demonstrated that mtDNA suffers more modification than nuclear DNA after exposure of cells to BaP or BaP DE (11,20). In isolated, whole mitochondria from BaP-treated rats, DNA synthesis is reduced but mitochondrial DNA polymerase activity increases, most likely as a feedback response to the high degree of mtDNA modification and decrease in ATP production (16). Treatment of cells with BaP inhibits mitochondrial DNA synthesis with the associated presence of 4–15 times more adducts per unit length of mtDNA than nuclear DNA (21). Recently, using a highly sensitive chemiluminescence immunoassay, levels of BaP DE adducts at dG were found to be 10-fold higher in mtDNA than nuclear DNA per unit length of DNA (22). Antioxidant anticancer treatment with N-acetylcysteine decreased the amount of mtDNA adducts in rats exposed to cigarette smoke (23), which contains PAH carcinogens, possibly by trapping the reactive DE metabolites. Thus, PAHs may exert a more profound effect in the cell on mtDNA relative to nuclear DNA. This may be due to the lipophilic character of these PAHs and the high ratio of lipid to DNA in mitochondria.
Mitochondrial DNA is replicated by an assembly of proteins and enzymes consisting of polymerase (pol) , single-stranded DNA binding protein, DNA helicase and a number of accessory proteins and transcription factors (24,25). In humans, pol consists of two subunits, a 140 kDa catalytic subunit containing DNA polymerase, exonucleolytic proofreading and dRP lyase activities and a smaller 55 kDa subunit that is a processivity factor with the ability to bind double-stranded DNA (26–28).
In the present study, translesion synthesis on DNA templates containing BaP or benzophenanthrene (BcPh) DE adducts by human recombinant pol was examined. We used oligonucleotide substrates, one in which a guanine (G) is modified and the other in which an adenosine (A) is modified, which had previously been examined in both mutagenesis (9) and polymerase bypass (29,30) studies. The human pol was able to incorporate correct and incorrect nucleotides opposite most of these adducts. The BaP and BcPh adducts caused significant blockage of replication with limited translesion synthesis past certain stereoisomers.
MATERIALS AND METHODS
Enzymes
The exonuclease-deficient form of human DNA pol (Exo–p140) was purified to homogeneity from baculovirus-infected insect cells as described (26). The recombinant human accessory subunit (p55) was purified to homogeneity from Escherichia coli and the heterodimeric forms of the polymerase were reconstituted as previously described (28).
Oligonucleotides
The G* templates containing a single trans-opened BaP DE trans R or trans S dG adduct (Fig. 1A) on the fourth nucleotide from 5'-end in the sequence context 5'-TTCG*AATCC TTCCCCC-3' (Fig. 1B) were synthesized as described previously (31,32). A* templates containing dA adducts (Fig. 1A), also on the fourth nucleotide from the 5'-end, in the sequence context 5'-CAGA*TTTAGAGTCTGC-3' (Fig. 1B) were synthesized as described: trans-opened BaP DE trans R or trans S dA adducts (33), cis-opened BaP DE cis R or cis S dA adducts (34) and cis-opened BcPh cis R or cis S dA adducts (35). All other oligonucleotides were purchased from Invitrogen Life Technologies, desalted and PAGE purified. The dG primer (5'-TCG ATC GAT CCA GGG GGA AGG A-3') for the G* templates was gel purified and labeled at the 5'-end with ATP by T4 polynucleotide kinase (36). The dG primer, the G* template containing either the BaP trans R or trans S dG adduct and a 12mer (5'-TGG ATC GAT CGA-3') were annealed in a 1:1.4:1.4 ratio to produce the substrate in Figure 1B. To construct substrates of suitable length, each adducted 16mer template and an additional 12mer were annealed to a 22mer primer (see Fig. 1B). Annealing was performed by heating the oligonucleotides to 90°C for 5 min followed by slow cooling to room temperature in 10 mM Tris–HCl, pH 7.5. Likewise, the dA primer (5'-TCG ATC GAT CCA GCA GAC TCT A-3') for the A* templates was gel purified and labeled at the 5'-end with ATP by T4 polynucleotide kinase. As above, the dA primer, each of the A* templates and the 12mer (5'-TGG ATC GAT CGA-3') were annealed in a 1:1.4:1.4 ratio to produce the substrates in Figure 1B. Control reactions for each set contained identical oligonucleotides except that the 16mer templates contained a normal dA or dG residue in place of the adducts.
DNA polymerase reactions
DNA pol reactions (10 μl final volume) contained final concentrations of 25 mM HEPES–KOH (pH 7.5), 5 mM MgCl2, various concentrations of the four dNTPs, 2 mM 2-mercaptoethanol, 50 μg/ml bovine serum albumin, 0.1 mM EDTA, 1 pmol 5'-32P-labeled 22mer previously annealed to 1.4 pmol 16mer template and 1.4 pmol 12mer (Fig. 1B). Reactions were started by addition of DNA pol (10 fmol for control substrates and 60 fmol for adducted substrates), either Exo–p140 alone or in complex with the accessory subunit (1.0:1.1 molar ratio). Samples were incubated for 5 min at 37°C. Reactions were stopped by addition of 10 μl of 95% aqueous formamide containing 25 mM EDTA and gel sequencing dye solution, followed by brief heating in a boiling water bath.
Incorporation analysis
Reaction products (4 μl) were loaded onto 15% polyacrylamide gels containing 7 M urea. Following electrophoresis, the gels were dried, exposed to a PhosphorImager screen and analyzed in a Storm 860 PhosphorImager (Molecular Dynamics). Radioactive bands were quantified with NIH Image software (version 1.62). Vmax and Km values were calculated using a non-linear Michaelis–Menten kinetic fit program from M.F. Goodman (USC). Misincorporation efficiencies were calculated from relative Vmax/Km values.
RESULTS
Human mitochondrial DNA pol has been tested for its ability to catalyze extension bypass of dG and dA adducts derived from two PAHs, as well as the specificity of nucleotide insertion opposite them using 22mer primer/28mer template complexes (see Materials and Methods). Structures of the adducts studied are shown in Figure 1A. These adducts are the BaP trans R and trans S dG adducts (in G* template–primer sets; Fig. 1B), the BaP trans R, trans S, cis R and cis S dA adducts and the BcPh cis S and cis R dA adducts (in A* template–primer sets). These adducts were copied by the exonuclease-deficient form of pol (Exo–p140) alone as well as in the complex with the pol accessory subunit, p55.
We first attempted to extend 10mer oligonucleotide primers annealed to the 16mer adducted and control templates. However, pol did not extend these short 16/10mer template–primers efficiently (<10% of primers extended), consistent with a requirement by the enzyme for a longer piece of DNA for optimal binding. We therefore synthesized a longer 22mer primer as well as a 12mer template extension and assembled the oligonucleotides as shown in Figure 1B. These longer template–primer sets were >3-fold more efficiently extended compared to the 10mer primers. We performed primer extension reactions with oligonucleotides in a running start reaction to reflect more accurately processive replication. These running start reactions were initiated by synthesis opposite either two T or two A residues in the template with a low concentration (1 μM) of either dATP or dTTP, respectively. The low dose of either dATP or dTTP was supplied to progress the polymerase to the site of the adduct. Incorporation opposite the adduct was tested by addition of each of the nucleoside triphosphates at high concentration. At least 1.0 mM dNTP was needed in order to detect incorporation opposite most of the adducts studied.
Pol -dependent replication of two diastereomeric BaP DE–dG adducts with and without the pol accessory subunit
Translesion synthesis by human pol with and without the accessory subunit was tested with the trans R and trans S diastereomers of the BaP DE–dG adducts using the first template–primer set in Figure 1B. As can be seen in lane 3 in each panel of Figure 2, the low concentration of dTTP was sufficient to fill the 2 nt gap prior to the site of the adduct, but did not cause any further incorporation or misincorporation opposite the adducts or the control template G. Addition of each nucleotide separately at high concentration is depicted in lanes 4–7 (Fig. 2A, Exo– p140 alone). With 1 mM each nucleoside triphosphate, pol easily incorporated the correct as well as the incorrect nucleotides on the control template. With the isolated catalytic subunit we even observed misincorporation of dGMP and extension of this misincorporation (lane 7 for no lesion), as well as incorrect elongation after a correctly incorporated dCMP residue (lane 6).
Figure 2. Single base insertion opposite normal dG and BaP trans R and trans S dG adducts in oligonucleotides. Reactions containing either Exo–p140 (A) or Exo–p140/p55 complex (B) were incubated for 5 min at 37°C. A partial sequence of the G* templates (Fig. 1B) is shown vertically on both panels of the figure, with the BaP-modified guanine indicated as G**. Lane 0, no dNTPs added; lane 4, all four dNTPs (each at 1 mM final concentration) added; lane T*, 1 μM dTTP (added to initiate a running start one base before the BaP-adducted guanine, G**); lanes A, T, C and G, 1 μM dTTP plus 1 mM one of the four dNTPs. The arrow (B) indicates the products of extension past the adduct.
The scenario for the action of pol on the substrates containing BaP DE–dG adducts was quite different. Here, pol incorporated mainly dAMP opposite the BaP DE adducts (Fig. 2A, lane 4), with a lesser efficiency of dGMP incorporation (Fig. 2A, lane 7). No correct incorporation of dCMP or incorrect incorporation of dTMP opposite these adducts was observed with the p140 subunit. The ability of the polymerase to extend past these adducts was tested by addition of all four dNTPs (lane 2 of each panel). As expected, the polymerase was easily able to extend synthesis to the end of the non-adducted template (Fig. 2A). Addition of all four dNTPs to reactions containing the BaP DE–dG adducted templates resulted in incorporation of only a single nucleotide residue opposite the adduct, similar to the results observed with either dGTP or dATP alone. Thus, the isolated catalytic subunit misincorporated either a dGMP or dAMP opposite the trans R and trans S adducts but could not extend these products.
Addition of the accessory p55 subunit (Fig. 2B) caused the polymerase to synthesize more product with the non-adducted template as well as with the BaP trans R and trans S dG adducted templates. With all four nucleotides present, pol with the accessory subunit inserted one or more nucleotides past the end of the template, most likely through a template dislocation intermediate or strand slippage mechanism that we previously documented for pol (37). The Exo– p140/p55 complex was able to incorporate all four nucleotides and extended past the resulting (mis)pair by at least one additional dAMP, dCMP or dGMP nucleotide on the control template with 1 mM each dNTP. With the BaP dG adducts, the p55 accessory subunit caused the polymerase to incorporate all of the nucleotides opposite the BaP DE–dG adducts, as compared to just dGMP or dAMP incorporation with the isolated catalytic subunit. In addition, a 1 nt translesion extension was observed with either 1 mM dATP or dGTP (Fig. 2B, lanes 4 and 7, arrow). In the presence of all four dNTPs (lane 2), the presence of p55 resulted in the 1 nt extension observed with either dATP or dGTP alone but failed to cause further extension.
Pol -dependent replication of four diastereomeric BaP DE–dA adducts with and without the pol accessory subunit
Translesion synthesis opposite BaP trans S, BaP trans R, BaP cis S and BaP cis R dA adducts was tested in a different sequence context, using the running start A* template–primer sets shown in Figure 1B. In these template–primer sets a low concentration of dATP was required to progress the polymerase to the site of the adduct in a processive synthesis. With the control template no significant amount of misincorporation of 1 μM dAMP by either the catalytic subunit alone or the complex containing the accessory subunit was observed (Fig. 3A and B, control template, lanes 3). With the addition of each of the four nucleoside triphosphates at high concentration (1 mM) to the control template we observed not only incorporation of the cognate dTMP into the primer but also the misincorporation of dAMP and dCMP. In addition extension of the A-C mispair by correct incorporation of dCMP opposite the next G was observed. Complete extension was observed when high concentrations of all four dNTPs were present. Qualitatively, the results were the same with and without the accessory subunit present.
Figure 3. Single base insertion opposite normal dA and BaP trans R, cis R, trans S and cis S dA adducts in oligonucleotides. Reactions containing either Exo–p140 (A) or Exo–p140/p55 complex (B) were incubated for 5 min at 37°C. A partial sequence of the A* templates (Fig. 1B) is shown vertically on both panels of the figure, with the BaP-modified adenine indicated as A**. Concentrations of the dNTPs and lane designations are as in Figure 2, except that each incubation contained 1 μM dATP to initiate a running start on the A* templates.
With the higher amount of pol needed to detect incorporation opposite the BaP DE–dA adduct we observed a slight amount of dAMP incorporation in the presence of the low 1 μM dATP needed to progress the polymerase to the site of the adduct. This amount of misincorporation with 1 μM dATP varied depending on adduct stereochemistry, but was generally low enough not to interfere with interpretation of the incorporation of the other nucleotides. In the presence of 1 mM dATP this misincorporation of dAMP opposite all four of the adducts was significantly increased. At high concentration the only other nucleotide to show significant incorporation relative to the background of dAMP was the cognate dTMP in the case of the BaP trans R and trans S dA adducts, while no significant dTMP incorporation was observed with the BaP cis R and cis S dA adducts. No dCMP or dGMP incorporation was observed with any of the BaP dA adducts. With all four nucleotides present only trace amounts of extension were observed on the BaP trans R and trans S dA templates and extension was not detected on the BaP cis R and cis S dA templates.
Upon addition of the p55 accessory subunit to these reactions, we observed enhanced incorporation with all of the BaP DE–dA adducts. The accessory subunit allowed correct incorporation of dTMP opposite the BaP trans R and trans S dA adducts, to about the same extent as the misincorporation of dA. There was no evidence for enhanced misincorporation of dCMP or dGMP opposite the trans adducts, since these bands (Fig. 3B) were the same as those observed with 1 μM dATP alone. The situation with the BaP cis R and cis S dA adducted templates was very different in the presence of the accessory subunit. Here, we observed primarily dAMP misincorporation as well as slight incorporation of dCMP and possibly dGMP, which were not observed in the absence of the p55 subunit. There was little or no enhanced incorporation of dTMP opposite the adduct in the presence of the accessory subunit. Thus the effect of the accessory subunit on incorporation opposite BaP DE–dA adducts varies with the stereoisomer.
Pol -dependent replication of two stereoisomeric BcPh DE–dA adducts with and without the pol accessory subunit
Incorporation and translesion synthesis on a running start template having the same sequence as that used with the BaP dA adducts (Fig. 1B) were also examined with BcPh cis R and cis S dA adducts. The incorporation by pol with and without the accessory subunit on the non-adducted template is shown as the control in Figure 4A and B, respectively. With the catalytic subunit alone, dAMP was the only nucleotide incorporated opposite either diastereomeric BcPh dA adduct, with more dAMP being misincorporated opposite the cis S diastereomer (Fig. 4A, lane 4). In the presence of all four dNTPs, incorporation (presumably of dA) opposite the adduct occurred, but no further translesion synthesis was observed (Fig. 4A, lane 2).
Figure 4. Single base insertion opposite normal dA and BcPh cis R and cis S dA adducts in oligonucleotides. Reactions containing either Exo–p140 (A) or Exo–p140/p55 complex (B) were incubated for 5 min at 37°C. A partial sequence of the A* template is shown vertically on both panels of the figure, with the BcPh-modified adenine indicated as A**. Concentrations of the dNTPs and lane designations are as in Figure 3.
Addition of the p55 accessory subunit to the BcPh-adducted template reactions allowed nucleotides other than dAMP to be incorporated. With the cis R diastereomer, all four nucleotides were incorporated. When all four dNTPs were present together, the pol complex was able to incorporate a small amount of nucleotide one base past the adduct, but no further extension was detected. With the BcPh cis S isomer, dCMP and dGMP but not the cognate dTMP were incorporated by the pol complex, and no further extension was detected.
Kinetics of nucleotide insertion opposite BaP and BcPh adducts by pol
To determine relative efficiency of incorporation and misincorporation by pol opposite the adducts, we performed kinetic analysis of primer extension reactions. As in our other experiments, exonuclease-deficient pol was used to avoid degradation of the primer by the proofreading activity and to simplify interpretation of the results. This strategy was imperative due to the relatively high amounts of enzyme required to detect incorporation opposite the adducts. With the G* template–primer sets, the running start reaction requires processive DNA synthesis by inserting two dTMPs (I1 and I2) prior to the target G* site for the correct or incorrect nucleotide (I3). From integration of the intensities of PhosphorImager scans of I1, I2 and I3, reaction velocities were determined and misinsertion efficiency (fins) was calculated as the ratio Vmax/Km for incorrect and correct nucleotides. Velocity was expressed as the ratio I3/I2 as described (38,39). The same approach used dATP to accomplish the running start with the A* template–primer sets (Fig. 1B).
For the kinetic studies with G* template–primer sets, the concentration of dTTP required to fill the I1 and I2 positions was lowered to minimize the misinsertion of dTMP opposite the dG adduct that we observed in Figure 2. Since incorporation opposite these adducts was very inefficient in the absence of the accessory subunit, only the kinetic parameters for the two-subunit complex are shown. Table 1 displays the apparent kinetic parameters for incorporation opposite the BaP trans S and trans R dG adducts. Steady-state assumptions allow the overall efficiency of each enzyme on a normal DNA template to be estimated as kcat/Km(dNTP), a parameter comparable to the pre-steady-state indicator of enzymatic efficiency kpol/Kd(dNTP) (40,41). With saturating template–primer concentrations the ratio kcat/Km(dNTP) does not change upon restriction of DNA synthesis (42), which is encountered by replication opposite damaged template bases, and kcat/Km(dNTP) remains a valid parameter for comparing insertion events by pol . Similarly, since steady-state parameters do not account for the expected differences in Kd(dNTP) with normal or damaged templates, comparisons of differential insertion by a given enzyme are best described as fins = (Vmax/Km)incorrect/(Vmax/Km)correct.
Table 1. Kinetics of insertion by the p140/p55 pol complex opposite the BaP trans R and BaP trans S dG adducts in the sequence 3'-...AAG*CTT-5'
Individual kinetic parameters are presented in the tables as Vmax/Km(dNTP) ratios and fins values. The kinetic values for the BaP trans R and trans S dG adducts (Table 1) corroborate the observations in Figure 2. Although fidelity of the exonuclease-deficient pol in the presence of the p55 accessory subunit on the non-adducted dG substrate was low, presumably due to the effects of the accessory subunit, the correct dCMP was still much more effectively inserted opposite a normal dG base than opposite either of the adducts. Incorporation of dCMP opposite either dG adduct was 200 000- to 400 000-fold less efficient than dCMP incorporation opposite the non-adducted template dG bases (compare Vmax/Km values between the non-adducted template and BaP-adducted templates). In fact, dCMP was the worst nucleotide incorporated, while dAMP and dGMP were most efficiently incorporated opposite the BaP dG adducts. The fins values indicate that dAMP was incorporated 4.3 to 4.7 times more readily than dCMP on the adducted templates, whereas dAMP was incorporated 100-fold less efficiently than dCMP on the non-adducted template. Pol showed little difference in incorporation efficiency of each nucleotide between the trans R and trans S isomers of the BaP DE–dG adducts (Table 1).
In contrast, the kinetics of nucleotide incorporation opposite the BaP DE–dA adducts by pol clearly revealed differences between the adduct stereoisomers (Table 2), especially in incorporation of dGMP and dCMP between the cis and trans adducts. With either the BaP trans R or trans S dA adduct we observed no detectable (>1% extended product) incorporation of dGMP or dCMP, whereas with either the cis R or cis S dA adduct we could readily detect and quantify the level of dGMP and dCMP incorporation. However, there was little difference between the BaP cis R and cis S dA adducts or between the BaP trans R and trans S dA adducts. Incorporation of the cognate dTMP opposite the BaP DE–dA adducts was between 7500- and 41 000-fold less efficient than dTMP incorporation on the non-adducted template. Incorrect dAMP was the most efficiently incorporated nucleotide opposite all four of the isomeric BaP DE–dA adducts, although its incorporation was still less efficient than that for dAMP misincorporation opposite the non-adducted dA template. As in the case of the dG adducts, incorporation of the correct nucleotide was more seriously hindered by an adduct than incorporation of an erroneous dAMP, resulting in increased misincorporation frequencies opposite the adducts.
Table 2. Kinetics of insertion by the p140/p55 pol complex opposite the BaP trans R, BaP trans S, BaP cis R and BaP cis S dA adducts in the sequence 3'-...TTA*GAC-5'
Incorporation opposite the BcPh cis R and cis S dA adducts (Fig. 1) was detected with each of the four dNTPs (Table 3). Vmax/Km ratios were quite similar for the BcPh cis dA adducts and their corresponding BaP analogs (within a factor of 3 or less except for dTMP incorporation opposite the BcPh cis R adduct, which was 6 times less efficient than opposite the corresponding BaP cis R adduct). Incorporation of the cognate dTMP was 106 000- and 41 000-fold less efficient on the BcPh cis R and cis S dA templates as compared to the non-adducted dA template, whereas misincorporation of dAMP on both BcPh templates was only 6- to 8-fold less efficient than dAMP misincorporation on the non-adducted control template. Incorrect dAMP was 40- and 9-fold more likely to be incorporated on the BcPh cis R and cis S templates, respectively, as compared to dTMP. This was in contrast to the control template, where dAMP is incorporated more than 300 times less efficiently than dTMP by pol . Among all the adduct-templated (mis)incorporations measured in this study, misincorporations of dAMP opposite the BcPh and BaP cis dA adducts were most efficient.
Table 3. Kinetics of insertion by the p140/p55 pol complex opposite the BcPh cis R and BcPh cis S dA adducts in the sequence 3'-...TTA*GAC-5'
DISCUSSION
Reports of translesion synthesis past DNA adducts by pol are limited. The ability of pol to synthesize past abasic sites has been studied, and the enzyme was found to stall the majority of the time (43). When pol does perform translesion synthesis past an abasic site, it prefers to incorporate dAMP opposite the site (43). Translesion synthesis by pol is readily accomplished opposite 7,8-dihydro-8-oxo-2'-deoxyguanosine (8-oxo-dG) template bases. Xenopus laevis pol incorporates dCMP 73% of the time while misincorporating dAMP 27% of the time opposite an 8-oxo-dG adduct (43). Translesion DNA synthesis past Pt–DNA adducts by human mitochondrial DNA polymerase has been demonstrated (44). Human pol displays a specificity for translesion synthesis past dien Pt–DNA adducts, with less synthesis past oxaliplatin and still less for cisplatin (44). While damage to mtDNA by BaP is well established (11,20–23,45), translesion synthesis past BaP adducts by pol has not previously been investigated. Here we have studied base incorporation and translesion synthesis opposite BaP DE–dG, BaP DE–dA and BcPh DE–dA adducts in DNA by human mitochondrial DNA polymerase with and without the p55 accessory subunit.
The catalytic subunit alone had weak ability to incorporate opposite all of the DE adducts studied and showed no translesion synthesis. Even in the presence of the p55 accessory subunit we observed only limited, one base extension beyond the BaP DE–dG adducts, and even weaker extension or no extension at all beyond dA adducts. For all the adducts studied, kinetics experiments in the presence of p55 indicated that the total incorporation of both correct and incorrect nucleotides opposite these adducts was decreased by 3–4 orders of magnitude relative to the unmodified control template. Thus these adducts present a substantial block to DNA replication in vitro. This blockage is consistent with the inhibition of mtDNA replication that was previously demonstrated with intact mitochondria upon treatment with BaP (16,21).
Pol containing its associated 3'5' proofreading exonuclease activity is highly accurate (37). The present study required use of exonuclease-deficient pol in order to avoid degradation of the primers, resulting in markedly reduced fidelity of replication for unmodified DNA. However, even in the absence of proofreading ability, pol still inserted predominantly the correct nucleotide opposite a normal dG or dA, with an error frequency of <10–3 for most cases. In contrast, pol with its accessory subunit incorporated all three incorrect nucleotides in preference to the correct dCMP opposite dG adducts. The nucleotides most frequently inserted opposite a dG adduct were the purine nucleotides dGMP and dAMP, with fins values of 3.7–4.7 for both nucleotides. This was a consequence of a dramatically reduced incorporation of the correct dCMP along with much smaller decreases in incorporation of the incorrect nucleotides. A similar effect was observed with the dA-adducted templates, with dAMP being incorporated in preference to the correct dTMP opposite all the dA adducts studied.
Pol is a family A DNA polymerase which includes such well-studied DNA polymerases as the E.coli pol I and T7 DNA polymerase. Thus, one might expect pol to exhibit the same pattern of translesion bypass with BaP DE adducts as E.coli pol I and T7 DNA polymerase. Studies with the Sequenase 2.0 version of T7 DNA polymerase demonstrated that BaP DE–dG adducts blocked base insertion opposite the adducts in vitro (46). On the basis of molecular modeling studies, preferential dAMP insertion opposite a BaP trans S dG adduct by the T7 DNA polymerase has been postulated to result from stabilizing interactions between the polymerase, DNA and dATP (47). With dA adducts, Sequenase was also shown to be very inefficient at nucleotide incorporation and, like pol , incorporated mostly an incorrect dAMP opposite both the trans R and trans S adducts (33). The response of A family pol to adducts is also reminiscent of that of the Y family bypass polymerase , which showed a strong preference for purine nucleotide misincorporation opposite BaP DE–dG adducts (30).
Mutations in mtDNA can induce a multitude of severe metabolic disorders and mitochondrial genetic disorders (48). Although translesion synthesis by pol in the present study was inefficient and was limited to a single base extension beyond BaP DE–dG adducts, our observation of enhanced misincorporation opposite the adducts suggests a mechanism for mutagenesis, should pol be able to replicate past these adducts in vivo. Another attractive, although indirect, mechanism exists for delayed phenotypic expression of mutations as a result of DNA damage induced by PAHs. Mitochondrial DNA is a multicopy genome, with as many as 10 000 mtDNA genomes per cell, and can exist in heteroplasmic form in which a low percentage of the genomes may contain mutations, either silent or deleterious. Depending on the tissue type, deleterious mutations in mtDNA can exist below a clinical threshold in which phenotypic symptoms do not show. Increasing the percentage of the genomes which carry such mutations above the respiratory threshold of that tissue causes tissue degeneration and mitochondrial disease. Although BaP damage to mtDNA is well established (11,20) and later studies have supported this finding (21–23,45), long-term mutagenicity of this damage to mtDNA has not been observed (49,50). This may be due to the fact that most of the adducted mitochondrial genomes cause a complete block in DNA replication and loss of the damaged genomes, consistent with the observation of a decrease in mtDNA replication and mtDNA copy number after BaP treatment without accompanying fixed mutations. Thus, the multicopy nature of mtDNA gives the cell the advantage of dispensing with damaged genomes instead of attempting to repair them. However, blockage of mtDNA replication and subsequent depletion in mtDNA may have another consequence on mtDNA heteroplasmy. Replication of the remaining mitochondrial genomes would amplify any mutations (from whatever source) in this surviving population. Thus, deleterious long-term consequences of adducts from BaP and other PAHs in mtDNA could result from pre-existing mutations which reach a physiologically significant level only after the mtDNA population has been depleted by PAH adduct-related DNA damage.
ACKNOWLEDGEMENTS
We thank Drs Matthew Longley and Kasia Bebenek for critical reading of this manuscript.
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